Tissue organoids and spheroids are powerful tools for many different applications, particularly in the area of drug screening. Yet, their widespread use is limited by a need for automated cell and tissue culture procedures and increased throughput. Here, we demonstrate that the BioAssembyBot (BAB) automation platform, with integrated BioStorageBot (BSB) and imaging system can perform tasks normally done by scientists with increased efficiency and reduced error. Using the BAB Hand| Pipette tool, BAB can precisely seed a Matrigel organoid suspension such that Matrigel domes are formed consistently in the center of each well plate. The BAB-automated process performed faster, with a higher success rate, and less organoid fragmentation than when the process is done by an experienced scientist. We then repeated this workflow with patient-derived colorectal cancer organoids (CRC PDOs) and performed a fully automated drug screen on cultured organoids. Image-based profiling performed on images that were automatically acquired was able to distinguish clear differences in patient response to anticancer drugs between two donors. Overall, we demonstrate the capability of the BioAssemblyBot automation platform to manage an entire organoid drug screening protocol with minimal human interaction. The system is user-friendly and may be adapted to a wide variety of workflows and applications, providing a solution to the need for organoid automation technology.   ### Competing Interest Statement MO, WO, VW, DE are or were employees of Core Life Analytics; VD, and HA were employees of Cellesce LTD; VS and AL are employed by Molecular Devices; and HS, JCH, SM, and JBH are or were employees of Advanced Solutions. JBH is a partner of Advanced Solutions.

Supplementary data including supplementary figures S1 to S6 and supplementary tables S1 to S3.

Supplementary information on the used materials and methods.

Growth hormone (GH) and insulin-like growth factor-1 (IGF1) play an important role in mammalian development, cell proliferation and lifespan. Especially in cases of tumor growth there is an urgent need to control the GH/IGF1 axis. In this study we screened a 38,480-compound library, and in two consecutive rounds of analogues selection, we identified active lead compounds based on the following criteria: inhibition the GH receptor (GHR) activity and its downstream effectors Jak2 and STAT5, and inhibition of growth of breast and colon cancer cells. The most active small molecule (BM001) inhibited both the GH/IGF1 axis and cell proliferation with an IC50 of 10-30 nM of human cancer cells. BM001 depleted GHR in human lymphoblasts. In preclinical xenografted experiments, BM001 showed a strong decrease in tumor volume in mice transplanted with MDA-MB-231 breast cancer cells. Mechanistically, the drug acts on the synthesis of the GHR. Our findings open the possibility to inhibit the GH/IGF1 axis with a small molecule.

AbstractNeuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2–dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2–dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Non-resistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2–dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2–dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax–idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies.

Life science experiments that employ automated technologies, such as high-content screens, frequently produce large datasets that require substantial amounts of preprocessing before analysis can be carried out. Standardization of this preprocessing becomes impossible as the dataset size increases if there are manual steps involved. Virtually no standards for preprocessing currently exist and few user-friendly tools are available that allow the cleaning of data files in a simple and transparent manner while also allowing for reproducibility. We demonstrate in a publicly available R package, PurifyR, how preprocessing steps can be streamlined and automated. PurifyR supports multithreading and the standardization of large-matrix preprocessing. These steps provide transparent and reproducible preprocessing for matrix-oriented datasets. The PurifyR package is open source and can be downloaded from github.

There has been an increase in the use of machine learning and artificial intelligence (AI) for the analysis of image-based cellular screens. The accuracy of these analyses, however, is greatly dependent on the quality of the training sets used for building the machine learning models. We propose that unsupervised exploratory methods should first be applied to the data set to gain a better insight into the quality of the data. This improves the selection and labeling of data for creating training sets before the application of machine learning. We demonstrate this using a high-content genome-wide small interfering RNA screen. We perform an unsupervised exploratory data analysis to facilitate the identification of four robust phenotypes, which we subsequently use as a training set for building a high-quality random forest machine learning model to differentiate four phenotypes with an accuracy of 91.1% and a kappa of 0.85. Our approach enhanced our ability to extract new knowledge from the screen when compared with the use of unsupervised methods alone.

BACKGROUND:Acute myeloid leukemia (AML) is a heterogeneous disease regarding morphology, immunophenotyping, genetic abnormalities, and clinical behavior. The overall survival rate of pediatric AML is 60% to 70%, and has not significantly improved over the past two decades. Children with Down syndrome (DS) are at risk of developing acute megakaryoblastic leukemia (AMKL), which can be preceded by a transient myeloproliferative disorder during the neonatal period. Intensification of current treatment protocols is not feasible due to already high treatment-related morbidity and mortality. Instead, more targeted therapies with less severe side effects are highly needed.PROCEDURE:To identify potential novel therapeutic targets for myeloid disorders in children, including DS-AMKL and non-DS-AML, we performed an unbiased compound screen of 80 small molecules targeting epigenetic regulators in three pediatric AML cell lines that are representative for different subtypes of pediatric AML. Three candidate compounds were validated and further evaluated in normal myeloid precursor cells during neutrophil differentiation and in (pre-)leukemic pediatric patient cells.RESULTS:Candidate drugs LMK235, NSC3852, and bromosporine were effective in all tested pediatric AML cell lines with antiproliferative, proapoptotic, and differentiation effects. Out of these three compounds, the pan-histone deacetylase inhibitor NSC3852 specifically induced growth arrest and apoptosis in pediatric AML cells, without disrupting normal neutrophil differentiation.CONCLUSION:NSC3852 is a potential candidate drug for further preclinical testing in pediatric AML and DS-AMKL.

Background CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. Results To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. Conclusions The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.

During embryonic development, axons extend over long distances to establish functional connections. In contrast, axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing CNS regeneration. Here, we performed one of the first miRNome-wide functional miRNA screens to identify miRNAs with robust effects on axon growth. High-content screening identified miR-135a and miR-135b as potent stimulators of axon growth and cortical neuron migration in vitro and in vivo in male and female mice. Intriguingly, both of these developmental effects of miR-135s relied in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon growth and regeneration. These results prompted us to test the effect of miR-135s on axon regeneration after injury. Our results show that intravitreal application of miR-135s facilitates retinal ganglion cell (RGC) axon regeneration after optic nerve injury in adult mice in part by repressing KLF4. In contrast, depletion of miR-135s further reduced RGC axon regeneration. Together, these data identify a novel neuronal role for miR-135s and the miR-135–KLF4 pathway and highlight the potential of miRNAs as tools for enhancing CNS axon regeneration. SIGNIFICANCE STATEMENT Axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing regeneration. By performing an miRNome-wide functional screen, our studies identify miR-135s as stimulators of axon growth and neuron migration and show that intravitreal application of these miRNAs facilitates CNS axon regeneration after nerve injury in adult mice. Intriguingly, these developmental and regeneration-promoting effects rely in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon regeneration. Our data identify a novel neuronal role for the miR-135–KLF4 pathway and support the idea that miRNAs can be used for enhancing CNS axon regeneration.

Abstract BACKGROUND: The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in the majority of all neuroblastomas. In previous preclinical studies, we have shown that treatment of BCL-2-dependent neuroblastoma with BCL-2 inhibitors leads to programmed cell death. These results have contributed to the initiation of a phase I trial to study the safety and pharmacokinetics of Venetoclax in children with relapsed or refractory neuroblastoma. AIM: The current study aims to identify and validate targeted combination strategies to prevent or overcome neuroblastoma resistance to Venetoclax. METHODS: Targeted drug candidates for combination treatment with Venetoclax were identified by high-throughput drug screening of non-resistant and Venetoclax-resistant BCL-2-dependent neuroblastoma cell lines. Top hits were subsequently tested more extensively in vitro and in vivo to validate the screening results. RESULTS: High-throughput drug screens identified the MDM2 inhibitor Idasanutlin as one of the strongest re-sensitizers for Venetoclax in Venetoclax-resistant BCL-2-dependent neuroblastoma cells with wild-type p53. Subsequent in vitro validation showed that Idasanutlin induced cyclin dependent kinase inhibitor 1 (p21)-mediated growth arrest in non-resistant neuroblastoma cells, but induced a BCL-2-associated X protein (BAX)-mediated apoptotic response in Venetoclax-resistant neuroblastoma cells in the presence of Venetoclax. In vivo combination of Venetoclax with Idasanultin resulted in a remarkably improved anticancer effect compared to single agent therapy, with very good partial and complete responses in BCL-2-dependent neuroblastoma xenografts. Combination screens additionally revealed that the clinically studied targeted inhibitors CUDC-907 (PI3K/HDAC), Prexasertib (CHK1) and Talazoparib (PARP1/2) improved the efficacy of Venetoclax more potently than ALK inhibitors in BCL-2-dependent neuroblastoma cells harboring ALK mutations. CONCLUSION: Our findings suggest that the clinical use of Venetoclax for the treatment of children with BCL-2-dependent neuroblastoma tumors can be improved by combination therapy with targeted inhibitors. The presence of additional neuroblastoma driving genomic events is not always predictive of the optimal combination strategy for BCL-2-dependent neuroblastoma subgroups. These findings should guide the design of combination Phase 2 trials of Venetoclax with other targeted compounds such as Idasanutlin, CUDC-907, Prexasertib and Talazoparib. Citation Format: M. Emmy M. Dolman, Laurel T. Bate-Eya, Lindy Vernooij, Bianca Koopmans, Lindy K. Alles, Anke H. Essing, Daphne Lelieveld, David A. Egan, Mark Kerstjens, Ronald W. Stam, Huib N. Caron, Jan J. Molenaar. Preclinical identification of Venetoclax combination strategies with Idasanutlin, CUDC-907, Prexasertib or Talazoparib for neuroblastoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2630.

Hepatic progenitor cells (HPCs) are adult liver stem cells that act as second line of defense in liver regeneration. They are normally quiescent, but in case of severe liver damage, HPC proliferation is triggered by external activation mechanisms from their niche. Although several important proproliferative mechanisms have been described, it is not known which key intracellular regulators govern the switch between HPC quiescence and active cell cycle. We performed a high-throughput kinome small interfering RNA (siRNA) screen in HepaRG cells, a HPC-like cell line, and evaluated the effect on proliferation with a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. One hit increased the percentage of EdU-positive cells after knockdown: dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Although upon DYRK1A silencing, the percentage of EdU- and phosphorylated histone H3 (pH3)-positive cells was increased, and total cell numbers were not increased, possibly through a subsequent delay in cell cycle progression. This phenotype was confirmed with chemical inhibition of DYRK1A using harmine and with primary HPCs cultured as liver organoids. DYRK1A inhibition impaired Dimerization Partner, RB-like, E2F, and multivulva class B (DREAM) complex formation in HPCs and abolished its transcriptional repression on cell cycle progression. To further analyze DYRK1A function in HPC proliferation, liver organoid cultures were established from mBACtgDyrk1A mice, which harbor one extra copy of the murine Dyrk1a gene (Dyrk+++). Dyrk+++ organoids had both a reduced percentage of EdU-positive cells and reduced proliferation compared with wild-type organoids. This study provides evidence for an essential role of DYRK1A as balanced regulator of S-phase entry in HPCs. An exact gene dosage is crucial, as both DYRK1A deficiency and overexpression affect HPC cell cycle progression.

Significance Wnt/β-catenin signaling is crucial for adult homeostasis and stem cell maintenance, and its dysregulation is strongly associated to cancer. Upon Wnt binding, Wnt receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The assembly and regulation of these signalosomes remains largely elusive. Here, we use internally tagged Wnt ligands as a tool to isolate and analyze the composition and regulation of endogenous Wnt receptor complexes. We identify a positive regulator of Wnt signaling that facilitates signalosome formation by promoting intramembrane receptor interactions. Our results reveal that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps and involves regulated intramembrane interactions.

Every cancer originates from a single cell. During expansion of the neoplastic cell population, individual cells acquire genetic and phenotypic differences from each other. Here, to investigate the nature and extent of intra-tumour diversification, we characterized organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells. Intra-tumour diversification of DNA methylation and transcriptome states also occurred; these alterations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. There were marked differences in responses to anticancer drugs between even closely related cells of the same tumour. The results indicate that colorectal cancer cells experience substantial increases in somatic mutation rate compared to normal colorectal cells, and that genetic diversification of each cancer is accompanied by pervasive, stable and inherited differences in the biological states of individual cancer cells.

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be (R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.

In this study, an experiment is conducted to measure the performance in speed and accuracy of interactive visualizations. A platform for interactive data visualizations was implemented using Django, D3, and Angular. Using this platform, a questionnaire was designed to measure a difference in performance between interactive and noninteractive data visualizations. In this questionnaire consisting of 12 questions, participants were given tasks in which they had to identify trends or patterns. Other tasks were directed at comparing and selecting algorithms with a certain outcome based on visualizations. All tasks were performed on high content screening data sets with the help of visualizations. The difference in time to carry out tasks and accuracy of performance was measured between a group viewing interactive visualizations and a group viewing noninteractive visualizations. The study shows a significant advantage in time and accuracy in the group that used interactive visualizations over the group that used noninteractive visualizations. In tasks comparing results of different algorithms, a significant decrease in time was observed in using interactive visualizations over noninteractive visualizations.

Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.

In response to microbial invasion, neutrophils release neutrophil extracellular traps (NETs) to trap and kill extracellular microbes. Alternatively, NET formation can result in tissue damage in inflammatory conditions and may perpetuate autoimmune disease. Intervention strategies that are aimed at modifying pathogenic NET formation should ideally preserve other neutrophil antimicrobial functions. We now show that signal inhibitory receptor on leukocytes-1 (SIRL-1) attenuates NET release by human neutrophils in response to distinct triggers, including opsonized Staphylococcus aureus and inflammatory danger signals. NET release has different kinetics depending on the stimulus, and rapid NET formation is independent of NADPH oxidase activity. In line with this, we show that NET release and reactive oxygen species production upon challenge with opsonized S. aureus require different signaling events. Importantly, engagement of SIRL-1 does not affect bacterially induced production of reactive oxygen species, and intracellular bacterial killing by neutrophils remains intact. Thus, our studies define SIRL-1 as an intervention point of benefit to suppress NET formation in disease while preserving intracellular antimicrobial defense.

High-content screening (HCS) can generate large multidimensional datasets and when aligned with the appropriate data mining tools, it can yield valuable insights into the mechanism of action of bioactive molecules. However, easy-to-use data mining tools are not widely available, with the result that these datasets are frequently underutilized. Here, we present HC StratoMineR, a web-based tool for high-content data analysis. It is a decision-supportive platform that guides even non-expert users through a high-content data analysis workflow. HC StratoMineR is built by using My Structured Query Language for storage and querying, PHP: Hypertext Preprocessor as the main programming language, and jQuery for additional user interface functionality. R is used for statistical calculations, logic and data visualizations. Furthermore, C++ and graphical processor unit power is diffusely embedded in R by using the rcpp and rpud libraries for operations that are computationally highly intensive. We show that we can use HC StratoMineR for the analysis of multivariate data from a high-content siRNA knock-down screen and a small-molecule screen. It can be used to rapidly filter out undesirable data; to select relevant data; and to perform quality control, data reduction, data exploration, morphological hit picking, and data clustering. Our results demonstrate that HC StratoMineR can be used to functionally categorize HCS hits and, thus, provide valuable information for hit prioritization.